Circadian rhythm is a fundamental property of all eukaryotic and some prokaryotic organisms, allowing the organism to adapt its cellular metabolism and physiology to the ~24 hr light-dark cycle of the earth. In mammals, circadian rhythm is generated by an autoregulatory transcription-translation feedback loop, the core components of which are CLOCK, BMAL1, Cryptochrome (CRY), and Period (PER) proteins. The heterodimeric CLOCK:BMAL1 transcriptional factor activates the transcription of genes Cry and Per, and is in turn inhibited by their protein products CRY and PER, ultimately creating rhythmic gene expression patterns. Circadian clock is estimated to affect the expression of at least 10% of all genes in human genome, thus influences many aspects of metabolism, physiology, and behavior. Currently, little is known about molecular interactions between CLOCK:BMAL1 and its negative regulator CRY and PER at biochemical and structural levels, and how such interactions modulate the activity of CLOCK:BMAL1. The goal of the proposed study is to elucidate molecular interactions between CLOCK:BMAL1, CRY, and PER, through rigorous in vitro biochemical, biophysical and structural investigations, and to delineate the structural mechanisms by which the transcriptional function of CLOCK:BMAL1 is modulated by PER and CRY. We will first generate various functionally relevant constructs of clock proteins based on functional and structural considerations (Aim 1). We have in hand the over-expressed and purified recombinant constructs of the structured regions of individual clock proteins and have determined the complex structure of CLOCK:BMAL1 in our preliminary studies. We will include in our investigation additional functional domains/motifs of clock proteins. CRY is the main repressor of CLOCK:BMAL1. We have demonstrated that CRY forms stable complex with CLOCK:BMAL1 in vitro. In Aim 2, we will determine the structure of CRY:CLOCK:BMAL1 complex to learn how CRY interacts with and represses the transactivation function of CLOCK:BMAL1. In the last Aim (Aim 3), we will investigate the interactions of PER with other clock components, CRY:PER and PER:CLOCK:BMAL1, by in vitro and in vivo protein-protein interaction assays, structure determination of functionally relevant complexes, as well as mutagenesis and functional analysis. We hope to learn the distinct role of CRY and PER in the mammalian clock mechanism and how they work together with CLOCK:BMAL1 to generate and maintain circadian rhythm in mammals.